Publications

Endosomes are nanoscale intracellular compartments that sort and recycle cell-surface receptors such as epidermal growth factor receptor-1 (EGFR1). Nanometer-scale interactions and coclustering of signaling proteins, cargo, and the membrane are critical to this process, yet direct 3D visualization has been hindered by the limited resolution of conventional and super-resolution microscopies. Here, we adapt expansion microscopy (ExM) to visualize and quantify nanoclusters of endosomal proteins in human retinal pigment epithelial (RPE-1) cells. We developed a 3D distortion analysis leveraging the Farneback optical-flow principle to detect anisotropies in hydrogel expansion, revealing under-expansion of cytoplasmic regions within ExM hydrogels and overestimation of size and distance measurements of small compartments such as endosomes. To calibrate ExM images of cytoplasmic regions containing endosomes, we introduced a self-assembling protein nanocage that reports the true local nanoscale expansion factor. To stimulate and visualize EGFR1 internalization and sorting, we applied a pulse-chase protocol with fluorescently tagged epidermal growth factor (EGF), fixed cells at 15 and 30 min, and subjected samples to 10-fold ExM and multiplexed 3D Airyscan microscopy to map cargo and EGFR1 relative to other endosomal proteins. A volume tracing pipeline was developed to visualize the changes in the labeled EGF and EGFR1 densities at the limiting membrane of the endosomes. These changes included enrichment of EGF and EGFR1 in the endosomal interior and accumulation of Rab5a near the limiting membrane during early endosome maturation. Together, this multiplexed 3D ExM toolkit provides a quantitative framework for visualizing and measuring small subcellular organelles at true molecular-scale resolution.

The nano-scale structure of human neuromuscular junctions (NMJs) has been difficult to assess using super-resolution imaging approaches due to practical and technical limitations associated with muscle biopsy specimens, as well as the high costs and specialized equipment required to perform super-resolution microscopy. To address this issue, we have developed and optimized expansion microscopy procedures suitable for use with human and mouse muscle tissues. We subsequently developed specialized morphological quantification approaches to compare nano-scale features of NMJs between species.

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, ‘calcium sparks’, determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.

Endosomes are nanoscale intracellular compartments that sort and recycle cell-surface receptors such as epidermal growth factor receptor-1 (EGFR1). Nanometer-scale interactions and coclustering of signaling proteins, cargo, and the membrane are critical to this process, yet direct 3D visualization has been hindered by the limited resolution of conventional and super-resolution microscopies. Here, we adapt expansion microscopy (ExM) to visualize and quantify nanoclusters of endosomal proteins in human retinal pigment epithelial (RPE-1) cells. We developed a 3D distortion analysis leveraging the Farneback optical-flow principle to detect anisotropies in hydrogel expansion, revealing under-expansion of cytoplasmic regions within ExM hydrogels and overestimation of size and distance measurements of small compartments such as endosomes. To calibrate ExM images of cytoplasmic regions containing endosomes, we introduced a self-assembling protein nanocage that reports the true local nanoscale expansion factor. To stimulate and visualize EGFR1 internalization and sorting, we applied a pulse-chase protocol with fluorescently tagged epidermal growth factor (EGF), fixed cells at 15 and 30 min, and subjected samples to 10-fold ExM and multiplexed 3D Airyscan microscopy to map cargo and EGFR1 relative to other endosomal proteins. A volume tracing pipeline was developed to visualize the changes in the labeled EGF and EGFR1 densities at the limiting membrane of the endosomes. These changes included enrichment of EGF and EGFR1 in the endosomal interior and accumulation of Rab5a near the limiting membrane during early endosome maturation. Together, this multiplexed 3D ExM toolkit provides a quantitative framework for visualizing and measuring small subcellular organelles at true molecular-scale resolution.

The nano-scale structure of human neuromuscular junctions (NMJs) has been difficult to assess using super-resolution imaging approaches due to practical and technical limitations associated with muscle biopsy specimens, as well as the high costs and specialized equipment required to perform super-resolution microscopy. To address this issue, we have developed and optimized expansion microscopy procedures suitable for use with human and mouse muscle tissues. We subsequently developed specialized morphological quantification approaches to compare nano-scale features of NMJs between species.

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, ‘calcium sparks’, determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.